Limiting the Persistence of a Chromosome BreakDiminishes Its Mutagenic PotentialNicole Bennardo1,2, Amanda Gunn1,2, Anita Cheng1, Paul Hasty3, Jeremy M. Stark1,2*1 Department of Cancer Biology, Division of Radiation Biology, Beckman Research Institute of the City of Hope, Duarte, California, United States of America, 2 City of HopeGraduate School of Biological Sciences, Duarte, California, United States of America, 3 Department of Molecular Medicine/Institute of Biotechnology, The University ofTexas Health Science Center at San Antonio, San Antonio, Texas, United States of AmericaAbstractTo characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring therepair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe theroles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcomeof such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products thatrestore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 39 exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI39 overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces thepotential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, wefind that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantialdeletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast,Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSBcauses a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find thatindividual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression ofI-SceI with Trex2.Citation: Bennardo N, Gunn A, Cheng A, Hasty P, Stark JM (2009) Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential. PLoSGenet 5(10): e1000683. doi:10.1371/journal.pgen.1000683Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of AmericaReceived June 25, 2009; Accepted September 15, 2009; Published October 16, 2009Copyright: ß 2009 Bennardo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by NIH grant RO1CA120954 to JMS. The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript.Competing Interest s: The authors have declared that no competing interests exist.* E-mail: [email protected] double-strand breaks (DSBs) can be repaired by anumber of mechanisms with a variety of mutagenic consequences[1]. In the context of ionizing radiation (IR) therapy orchemotherapy that utilizes DSB-inducing agents, such DNAdamage in non-tumor cells could result in oncogenic mutationsthat cause secondary malignancies [2]. Thus, characterizing thefactors and pathways that influence DSB repair will be importantto develop therapeutic approaches that may limit the risk ofsecondary tumors, and to understand the etiology of genomerearrangements associated with primary cancer development.DSB repair pathways show a varying propensity for genetic loss.A relatively precise form of repair is homology-directed repair(HDR) that uses the identical sister chromatid as a template forRad51-mediated strand invasion and nascent DNA synthesis [1].In contrast, end-joining (EJ) pathways are variably mutagenic,depending on the extent of end-processing and the fidelity of end-pairing. For instance, EJ via the V(D)J recombination nonhomol-ogous end-joining (NHEJ) machinery has the potential to beprecise, especially when DSB ends can be ligated withoutsignificant processing [3]. However, Ku-independent EJ (Alterna-tive-NHEJ, Alt-NHEJ) often leads to deletion mutations, which arepredominantly associated with short stretches of homology(microhomology) at repair junctions [4,5]. Similar to Alt-NHEJis single-strand annealing (SSA), which also causes deletions withhomology at repair junctions, but involves extensive regions ofhomology [6]. In addition, for each of these pathways, loss ofcorrect end-pairing during the repair of multiple simultaneousDSBs can lead to chromosomal rearrangements. For instance, EJbetween distal ends of two tandem DSBs (Distal-EJ) results in lossof the chromosomal segment between the DSBs.To characterize the genetic factors that influence these pathways,one approach involves analyzing repair of site-specific DSBs inmammalian cells, such as those generated by the rare-cuttingendonuclease I-SceI. For instance, using this approach, HDR, SSA,and Alt-NHEJ were shown to be promoted by CtIP and Nbs1[7–10], which are factors implicated in the formation of ssDNA viaend resection [9,11]. As well, the strand exchange factors Rad51/Brca2 were found to promote HDR and suppress SSA [12,13], anda number of additional genetic factors have been found to promoteHDR [14]. Other studies have addressed the influence of factorsinvolved in NHEJ during V(D)J recombination, including Ku andXrcc4-Ligase IV. For example, Ku/Xrcc4-deficient cells showhigher HDR [15], and Ku-deficient cells show elevated SSA andAlt-NHEJ [5]. In addition, Ku and Xrcc4 have been shown topromote EJ that restores the I-SceI site, measured as EJ betweendistal ends of two tandem I-SceI-induced DSBs (S+DEJ) [16,17].To further address the process of DSB repair pathway choice inmammalian cells, we have developed this two-part study. In thePLoS Genetics | www.plosgenetics.org 1 October 2009 | Volume 5 | Issue 10 | e1000683first part, we provide a detailed characterization of the roles ofErcc1, Msh2, Nbs1, Xrcc4, and Brca1 during individual repairevents. From these studies, we provide evidence that individualgenetic factors may not be specific for particular pathways of repair,but rather promote a mechanistic step that is
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